Mechanosensitivity is required for the efficient and effective processing of intestinal content. The overall objective of this proposal is to identify in human intestinal smooth muscle cells and in interstitial cells of Cajal (ICC) the mechanisms and physiological and pathophysiological relevance of mechanosensitivity. Our preliminary data Ca2+, suggest that mechanosensitive (MS) Ca2+ channels in human intestinal smooth muscle regulate Ca2+ influx in response to mechanical stimuli and that their expression is altered in disease states. Our preliminary data also suggest that human intestinal smooth muscle and ICC express a novel Na+ channel that is MS and that motility may be altered in patients with mutations in the Na+ channel or in patients with altered expression of the channel. Based on this work we have generated the novel hypothesis that intestinal contractility is regulated by MS L-type Ca2+ channels expressed in intestinal smooth muscle and MS Na+ channels expressed in intestinal smooth muscle and ICC and that disturbed mechanosensitivity may lead to motility disorders. The proposal has two specific aims. Our first aim will focus on the physiological and pathophysiological role of MS L-type Ca 2+ channels in intestinal smooth muscle by directly testing the hypotheses that: a) activation of MS L-type Ca2+ channels by stretch increases Ca2+ entry and contraction; b) the expressed L-type Ca2+ channel is MS; and c) upregulation of MS L-type Ca2+ channels in obstruction results in increased contractile force. Our second aim will focus on the physiological and pathophysiological role of MS Na+ channels in human intestinal smooth muscle cells and ICC by directly testing the hypotheses that: a) MS Na+ channels are present in smooth muscle cells and ICC and that mechanoactivation increases Na+ entry and regulates the membrane potential; b) the mammalian gene encoding the alpha subunit of the Na+ channel is SCN5A; and c) altered function of MS Na+ channels may contribute to the development of intestinal pseudo-obstruction. The studies will employ patch clamp techniques on freshly dissociated human smooth muscle cells and ICC, muscle strip techniques, optical laser tweezers, laser capture micro-dissection, immunohistochemistry, single cell PCR and standard molecular biology techniques to answer the questions raised. We are now uniquely poised to pursue our long term goal of providing new information of the role of MS ion channels in the regulation of intestinal contractility in both health and disease states. This work has the potential of identifying the pathophysiology of a subset of motility disorders such as intestinal pseudo-obstruction as well as provide novel therapeutic strategies.